title: Clinical and molecular characterization of a cohort of patients with novel nucleotide alterations of the Dystrophin gene detected by direct sequencing.
abstract: BACKGROUND
  Duchenne and Becker Muscular dystrophies (DMD/BMD) are allelic disorders caused by mutations in the dystrophin gene, which encodes a sarcolemmal protein responsible for muscle integrity. Deletions and duplications account for approximately 75% of mutations in DMD and 85% in BMD. The implementation of techniques allowing complete gene sequencing has focused attention on small point mutations and other mechanisms underlying complex rearrangements.
  
  
  METHODS
  We selected 47 patients (41 families; 35 DMD, 6 BMD) without deletions and duplications in DMD gene (excluded by multiplex ligation-dependent probe amplification and multiplex polymerase chain reaction analysis). This cohort was investigated by systematic direct sequence analysis to study sequence variation. We focused our attention on rare mutational events which were further studied through transcript analysis.
  
  
  RESULTS
  We identified 40 different nucleotide alterations in DMD gene and their clinical correlates; altogether, 16 mutations were novel. DMD probands carried 9 microinsertions/microdeletions, 19 nonsense mutations, and 7 splice-site mutations. BMD patients carried 2 nonsense mutations, 2 splice-site mutations, 1 missense substitution, and 1 single base insertion. The most frequent stop codon was TGA (n=10 patients), followed by TAG (n=7) and TAA (n=4). We also analyzed the molecular mechanisms of five rare mutational events. They are two frame-shifting mutations in the DMD gene 3'end in BMD and three novel splicing defects: IVS42: c.6118-3C>A, which causes a leaky splice-site; c.9560A>G, which determines a cryptic splice-site activation and c.9564-426 T>G, which creates pseudoexon retention within IVS65.
  
  
  CONCLUSIONS
  The analysis of our patients' sample, carrying point mutations or complex rearrangements in DMD gene, contributes to the knowledge on phenotypic correlations in dystrophinopatic patients and can provide a better understanding of pre-mRNA maturation defects and dystrophin functional domains. These data can have a prognostic relevance and can be useful in directing new therapeutic approaches, which rely on a precise definition of the genetic defects as well as their molecular consequences.
authors.affiliation: Dino Ferrari Centre, Department of Neurological Sciences, University of Milan, IRCCS Foundation Cà Granda, Ospedale Maggiore Policlinico, Milan, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Magri
authors.forename: Roberto
authors.initials: R
authors.lastname: Del Bo
authors.forename: Maria G
authors.initials: MG
authors.lastname: D'Angelo
authors.forename: Alessandra
authors.initials: A
authors.lastname: Govoni
authors.forename: Serena
authors.initials: S
authors.lastname: Ghezzi
authors.forename: Sandra
authors.initials: S
authors.lastname: Gandossini
authors.forename: Monica
authors.initials: M
authors.lastname: Sciacco
authors.forename: Patrizia
authors.initials: P
authors.lastname: Ciscato
authors.forename: Andreina
authors.initials: A
authors.lastname: Bordoni
authors.forename: Silvana
authors.initials: S
authors.lastname: Tedeschi
authors.forename: Francesco
authors.initials: F
authors.lastname: Fortunato
authors.forename: Valeria
authors.initials: V
authors.lastname: Lucchini
authors.forename: Matteo
authors.initials: M
authors.lastname: Cereda
authors.forename: Stefania
authors.initials: S
authors.lastname: Corti
authors.forename: Maurizio
authors.initials: M
authors.lastname: Moggio
authors.forename: Nereo
authors.initials: N
authors.lastname: Bresolin
authors.forename: Giacomo P
authors.initials: GP
authors.lastname: Comi
mesh_terms: D000293:Adolescent; D000328:Adult; D000483:Alleles; D017398:Alternative Splicing; D002648:Child; D002675:Child, Preschool; D018389:Codon, Nonsense; D018388:Codon, Terminator; D015331:Cohort Studies; D016189:Dystrophin / Q000235:genetics*; D016368:Frameshift Mutation; D006801:Humans; D008875:Middle Aged; D020388:Muscular Dystrophy, Duchenne / Q000175:diagnosis / Q000235:genetics*; D016254:Mutagenesis, Insertional; D020125:Mutation, Missense; D010641:Phenotype; D017422:Sequence Analysis, DNA
chemical_list: D018389:Codon, Nonsense; D018388:Codon, Terminator; D016189:Dystrophin